The Pro-Oxidant Activity of Red Wine Polyphenols Induces an Adaptive Antioxidant Response in Human Erythrocytes

Abstract

The protective effect of dealcoholized red wine on human health has been partially associated with its polyphenolic components, suggesting that the pool of polyphenols, including flavonoids and anthocyanins, can be responsible for the functional effects of this beverage. We hypothesize a new role of red wine polyphenols (RWp) in modulating the antioxidant potential of erythrocytes, protecting them against oxidative stress. We previously demonstrated that RWp activated the Plasma Membrane Redox System (PMRS), which is involved in neutralizing plasma free radicals. Here, we investigated the underlying mechanism triggered by RWp in the activation of PMRS via the involvement of GSH. Hence, treatment of human erythrocytes with RWp (73 μg/mL Gallic Acid Equivalents) increased GSH intracellular concentration, which depends upon the activation of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD), whose enzymatic activities increase of about 30% and 47%, respectively. Changes in the GSH pathway induced by RWp were associated with a slight but significant increase in reactive oxygen species (ROS). We conclude that the pro-oxidant effect of RWp promoted an adaptive stress response in human erythrocytes, which enhances their antioxidant defense.

Keywords: PMRS; adaptive response; antioxidant; erythrocytes; red wine polyphenols.

Figure 1

ROS production induced by RWp. Erythrocytes isolated from volunteers (n = 5) were incubated with RWp (73 μg/mL GAE) at the indicated times. Intracellular ROS levels were determined by using DCFDA/DCF fluorescent method and the results were expressed as % DCF, as described in “Materials and Methods”. Bar graphs represent the mean of 5 independent determinations, each performed in duplicate, ± SE. Symbols (*) in the graph indicate statistical significance: * p < 0.05 and ** p < 0.01 respect to untreated cells (CTRL).

Figure 2

Activation of GSH-dependent enzymes by RWp. Erythrocytes from volunteers (n = 7) were incubated with RWp (73 μg/mL GAE) at the indicated times. (a) GR activity of erythrocytes, expressed as nmol/min/mL erythrocytes. (b) G6PD activity of erythrocytes, expressed as nmol/min/mL erythrocytes. Bar graphs represent the mean of 7 independent determinations, each performed in duplicate, ± SE. Symbols (*) in the graph indicate statistical significance: *** p < 0.001 respect to untreated cells (CTRL); # p < 0.05 significance between 1 and 2 min of incubation with RWp.

Figure 3

Activation of antioxidant enzymes by RWp. Erythrocytes from volunteers (n = 5) were incubated with RWp (73 μg/mL GAE) at the indicated times. (a) SOD activity of erythrocytes, expressed as U/mL erythrocytes. (b) CAT activity of erythrocytes, expressed as % inhibition. Bar graphs represent the mean of 5 independent determinations, each performed in duplicate, ± SE. Symbols (*) in the graph indicate statistical significance: * p < 0.05 and ** p < 0.01 respect to untreated cells (CTRL).

Figure 4

Activation of PMRS by RWplm. Erythrocytes from volunteers (n = 5) were incubated in presence or absence of RWp and RWplm. (a) PMRS activity of erythrocytes incubated for 10 min with RWp and RWplm (73 μg/mL GAE, 27 μg/mL GAE, respectively). (b) PMRS activity of erythrocytes incubated for 10 min with the same concentration of RWp and RWplm (73 μg/mL GAE). PMRS activity was expressed as pmol ferrocyanide/10⁶ erythrocytes/min as reported in “Materials and Methods”. Bar graphs represent the mean of 5 independent determinations, each performed in duplicate, ± SE. Symbols (*) in the graph indicate statistical significance: *** p < 0.001 respect to untreated cells (CTRL).

Figure 5

PMRS activity of erythrocytes treated with RWplm, Kplm and Tplm. Erythrocytes from volunteers (n = 5) were incubated with RWplm, Kplm and Tplm (0.12 mg/mL, w/V) for 10 min. Results were expressed as pmol ferrocyanide/10⁶ erythrocytes/min as reported in “Materials and Methods”. Bar graphs represent the mean of 5 independent determinations, each performed in duplicate, ± SE. Symbols (*) in the graph indicate statistical significance: *** p < 0.001 respect to untreated cells (CTRL).

Figure 6

GSH intracellular concentration in erythrocytes. Erythrocytes from volunteers (n = 5) were incubated with the Scheme 73. and 27 μg/mL GAE, respectively) for 10 min. Results were expressed in terms of μM GSH as reported in “Materials and Methods”. Bar graphs represent the mean of 5 independent determinations, each performed in duplicate, ± SE. Symbols (*) in the graph indicate statistical significance: * p < 0.05 respect to untreated (CTRL).

Figure 7

Original graphical scheme representing the proposed mechanisms underlying the protective effect of RWp (see text for description).